Signal peptidase is a membrane-bound proteolytic enzyme that cleaves signal peptides from secretory proteins targeted to the lumen of the endoplasmic reticulum. The enzyme activity is associated with a complex of from two to five integral membrane proteins with their globular domains on the lumenal side of the endoplasmic reticulum. The enzyme is essential for viability in yeast and is presumed to be an essential gene of all eukaryotic organisms. Mutations in secretory proteins affecting cleavage of the signal peptide by signal peptidase have been described that have been described that result in hemophilia (coagulation factor X), familial neurohypophyseal diabetes insipidus (vasopressin), and a form of familial hypoparathyroidism (parathyroid hormone). Many other such inherited defects probably exist but have not been recognized. Because of the central role of this enzyme in protein biosynthesis and secretion, it is important to fully understand its function and role in protein translocation into the endoplasmic reticulum. The catalytic mechanism of microsomal signal peptidase appears to be novel and many define a new mechanistic proteinase class. The amino acid sequences of the known signal peptidase proteins are unrelated to any of the well established proteinase families. The major goal of this project is to define the structural and functional properties of the signal peptidase of the endoplasmic reticulum of animal cells. The project has three specific aims: 1) to identify catalytically functional amino acid residues; 2) to define the extended substrate cleavage specificity; and 3) to identify the protein components of microsomal signal peptidase that are essential for signal peptide processing. Specific, irreversible inhibitors of signal peptidase activity will be used to localize and identify amino acid residues required for catalysis. Modified proteins and peptide fragments of inhibited peptidase will be purified by high performance liquid chromatography and analyzed by animo acid sequence analysis and/or electrospray ionization mass spectrometry. Additional, more highly specific inhibitors of signal peptidase will be prepared. A model preprotein signal peptidase substrate will be modified by site-directed mutagenesis and used to reveal the structural determinants of signal peptides that define the extended substrate binding site. Microsomal signal peptidase will be expressed in tissue culture and bacterial cells to identify the individual protein components necessary and sufficient for recognition and cleavage of signal peptides in vivo. Structural and mechanistic studies will be greatly facilitated by the ability to produce and purify an enzymatically active, recombinant signal peptidase complex. Achievement of this goal will permit precise determination of the mechanism of action of this unusual membrane- bound proteinase.